HISTIDINE-52 IS A CRITICAL RESIDUE FOR RAPID FORMATION OF CYTOCHROME-C PEROXIDASE COMPOUND-I
HISTIDINE-52 IS A CRITICAL RESIDUE FOR RAPID FORMATION OF CYTOCHROME-C PEROXIDASE COMPOUND-I
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DOI：
10.1021/bi00088a035
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发表时间：
1993-09-21
期刊：
影响因子：
2.9
通讯作者：
KRAUT, J
中科院分区：
文献类型：
--
作者：
ERMAN, JE;VITELLO, LB;KRAUT, J
报道了一种克隆的细胞色素c过氧化物酶[CcP（MI）]的突变体的晶体结构和与过氧化氢的反应性，其中保守的远端His（His-52）被Leu取代。H52 L酶与过氧化物的反应在0.1M磷酸盐缓冲液和其中硝酸盐用于调节离子强度的缓冲液中作为pH的函数进行检查。在含磷酸盐和硝酸盐的缓冲液中，H52 L与过氧化物反应的pH非依赖性双分子速率常数分别为731 +/- 44和236 +/- 14 M-1 s-1。这表示在可比条件下，相对于CcP（MI）母体，速率降低了10（5）倍。单晶衍射研究表明，没有显着变化的结构或血红素结合位点的可及性所造成的突变。相反，突变仅在残基52和附近的活性位点水分子处引起显著的结构变化。H52 L酶与过氧化物的残余反应性是pH和缓冲液依赖性的。在含硝酸盐的缓冲液中，与过氧化物反应的表观双分子速率常数随pH值降低而降低;反应性的丧失与表观pK（A）= 4.5的基团的质子化相关。该基团的质子化导致与过氧化物的反应性丧失。这与CcP（MI）亲本酶以及已检查的所有其他突变体相反，其中反应性的丧失与表观pK（A）= 5.4的酶基团的质子化相关。在磷酸盐缓冲液中，表观双分子速率常数随着pH值的降低而增加，表观pK（A）= 4.2的基团的质子化使表观双分子速率常数增加至（1.2 +/- 0.7）x 10（4）M-1 S-1。结果表明，His-52对于CcP（MI）与过氧化物的快速反应至关重要;当该残基被Leu取代时，酶与过氧化物的反应性与高铁肌红蛋白的反应性相当。结果还表明，在CcP（MI）中的His-52的质子化导致在含硝酸盐的缓冲液中在酸性pH下观察到的酶失活。含磷酸盐和硝酸盐的缓冲液对H52 L与过氧化物反应的影响提供了证据，表明该酶可以受到特定离子效应的影响，这些离子效应不涉及His-52质子化状态的变化。
The crystal structure and reactivity with hydrogen peroxide are reported for a mutant of a cloned cytochrome c peroxidase [CcP(MI)], in which the conserved distal His (His-52) is replaced with Leu. The reaction of the H52L enzyme with peroxide was examined as a function of pH in 0.1 M phosphate buffers and buffers in which nitrate was used to adjust the ionic strength. The pH-independent bimolecular rate constant for the reaction of H52L with peroxide was 731 +/- 44 and 236 +/- 14 M-1 s-1 in phosphate and nitrate-containing buffers, respectively. This represents a 10(5)-fold decrease in rate relative to the CcP(MI) parent under comparable conditions. Single-crystal diffraction studies showed that no dramatic changes in the structure or in the accessibility of the heme binding site were caused by the mutation. Rather, the mutation caused significant structural changes only at residue 52 and the nearby active-site water molecules. The residual reactivity of the H52L enzyme with peroxide was pH- and buffer-dependent. In nitrate containing buffer, the apparent bimolecular rate constant for the reaction with peroxide decreased with decreasing pH; the loss of reactivity correlated with protonation of a group with an apparent pK(A) = 4.5. Protonation of the group caused a loss of reactivity with peroxide. This is in contrast to the CcP(MI) parent enzyme, as well as all other mutants that have been examined, where the loss of reactivity correlates with protonation of an enzyme group with an apparent pK(A) = 5.4. In phosphate buffer, the apparent bimolecular rate constant increased with decreasing pH, and protonation of a group with an apparent pK(A) = 4.2 increased the apparent bimolecular rate constant to (1.2 +/- 0.7) x 10(4) M-1 S-1. The results demonstrate that His-52 is crucial for the rapid reaction of CcP(MI) with peroxide; when this residue is replaced with Leu, the reactivity of the enzyme with peroxide is comparable to that of metmyoglobin. The results also suggest that protonation of His-52 in CcP(MI) causes the observed inactivation of the enzyme at acidic pH in nitrate-containing buffers. The effects of phosphate and nitrate-containing buffers on the reaction of H52L with peroxide provide evidence that the enzyme can be influenced by specific ion effects that do not involve changes in the protonation state of His-52.